I propose to continue our ongoing studies of the mechanisms involved in regulation of neuronal electrical activity. A detailed understanding of such regulation will be necessary for the rational design of treatments for disorders of the nervous system. Specifically, we will investigate the molecular mechanisms by which the intracellular second messengers cyclic AMP (cAMP), cyclic GMP (cGMP) and calcium (Ca++) interact to regulate the overall electrical state of indentified Aplysia neurons. The large size and ready identifiability of these neurons allow one to carry out multidisciplinary studies at the level of single indentified cells. The following approaches will be used: (1)Electrophysiological characterization of specific ion channels using intracellular recording and voltage clamp methodology. (2)Injection of specific molecular probes directly into individual nerve cells, to examine their effects on ion channels. The probes to be used will include purified protein kinases, calmodulin, and specific antibodies against protein kinases and calmodulin. (3)Biochemical characterization of cAMP-, cGMP- and Ca++ dependent protein kinases, and the substrate proteins which they phosphorylate. This will include he measurement of protein phosphorylation within single living cells by isotope injection techniques, to determine the relationship between phosphorylation and the electrical state of the cell. The long-term objective of these combined biochemical/physiological studies is to understand in molecular terms the physiological regulation of neuronal electrical activity.